Nucleotide-binding properties of kinase-deficient epidermal-growth-factor-receptor mutants.
The nucleotide-binding properties of wild-type epidermal- growth-factor (EGF)-receptor protein tyrosine kinase (PTK) and EGF-receptor mutants with site-specific amino acid substitutions known to attenuate protein kinase activity were analysed by a fluorescence competition assay employing the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. Binding affinities for ATP and Mn.ATP complex were determined for the PTK domains of the wild-type and two mutant proteins. Surprisingly, mutation of the highly conserved Lys-721 residue in the nucleotide-binding site of the EGF- receptor PTK domain did not abolish ATP and Mn.ATP binding, although the binding affinity for the Mn.ATP complex was significantly reduced. A second kinase-inactivating mutation that targeted the highly conserved Asp-813 residue had little effect on the nucleotide-binding properties of the EGF-receptor PTK domain. These results indicated that the principle effect of these two kinase-inactivating amino acid substitutions is not to block nucleotide binding, but is instead an inhibition of the phospho-transfer reaction.
Cheng
K
K
Department of Pharmacology, University of Iowa College of Medicine, Iowa City, IA 52242-1109, USA.
Koland
J G
JG
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid; D002384:Catalysis; D002942:Circular Dichroism; D003600:Cytosol; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine; D008345:Manganese / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D009711:Nucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D008345:Manganese; D014364:Tryptophan; D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D008239:Lysine